Modified Zuo Gui Wan Ameliorates Ovariectomy-Induced Osteoporosis in Rats by Regulating the SCFA-GPR41-p38MAPK Signaling Pathway

Changheng Song,1,2,* Qiqi Yan,1,* Yujie Ma,3 Pei Li,1 Ying Yang,1 Yuhan Wang,1 Wenjie Li,1 Xinyu Wan,1 Yubo Li,1 Ruyuan Zhu,1 Haixia Liu,1 Zhiguo Zhang1 1Institute of Basic Theory for Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, People’s Republic of China; 2Department of Endocrinology, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, People’s Republic of China; 3Science and Technology Innovation Center, Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Haixia Liu; Zhiguo Zhang, Institute of Basic Theory for Chinese Medicine, China Academy of Chinese Medical Sciences, No. 16, DongzhimenNei Nanxiaojie Street, Dongcheng District, Beijing, 100700, People’s Republic of China, Email [email protected]; [email protected]: Modified Zuo Gui Wan (MZGW) was a combination of Zuo Gui Wan and red yeast rice used for treating osteoporosis (OP), but its mechanism remains unclear. We aimed to validate the anti-OP effect of MZGW and explore its underlying mechanism.Methods: An ovariectomy (OVX) rat model in vivo and a RANKL-induced osteoclasts (OCs) model in vitro were established. Key active ingredients in MZGW high dose (MZGW-H) group were detected by UPLC-MS/MS. Micro-CT scans and histomorphology analysis were performed in OVX rats. 16S rRNA gene sequencing was performed to investigate the relationship between the anti-OP effect of MZGW-H and intestinal flora. CCK-8 assay was applied to examine the optimal concentration of Modified Zuo Gui Wan drug serum (MZGW-DS) on osteoclasts. The qRT-PCR and Western blotting were utilized to explore the potential anti-OP pathway of MZGW, namely the SCFA-GPR41-p38MAPK signaling pathway. GPR41 was knocked down to further reverse to verify whether the pathway was the key pathway for MZGW-DS to exert its inhibitory effect on osteoclasts.Results: The three main blood components, Ferulic acid, L-Ascorbic acid and Riboflavin, were examined mainly by UPLC-MS/MS. 16S rRNA gene sequencing showed that MZGW-H changed the metabolism of SCFAs. In vivo studies verified that MZGW-H ameliorated microstructure damage, improved histological changes and reduced TRAP, BALP, and BGP in OVX rats by regulating the SCFA-GPR41-p38MAPK signaling pathway. CCK-8 revealed that 5% MZGW-DS group was the most optimal concentration of MZGW-DS to inhibit osteoclast differentiation. In vitro, MZGW-DS was better than peripheral blood concentration of SCFAs in inhibiting osteoclasts. After the knockout of GPR41, MZGW-DS could not inhibit the expression of osteoclast-related protein (CTSK and NFATc1) via SCFA-GPR41-p38MAPK signaling pathway.Conclusion: MZGW-H effectively ameliorates OVX-induced osteoporosis partially achieved by increasing SCFAs metabolism and modulating the SCFA-GPR41-p38MAPK signaling pathway. Keywords: osteoporosis, short chain fatty acids, SCFA-GPR41-p38MAPK signaling pathway, osteoclasts, modified Zuo Gui Wan